1 Field of the Invention
The present invention concerns a process for the production of glycoproteins in mammalian cell culture. More specifically, the invention provides a process for producing glycoproteins in mammalian cells that results in enhanced occupancy of an N-linked glycosylation site occupied only in a fraction of a glycoprotein. A process for increasing the fraction of Type I tissue plasminogen activator (t-PA) in a mammalian cell culture is specifically disclosed.
2. Description of Related Disclosures and Technology
Glycoproteins, many of which have been produced by techniques of recombinant DNA technology, are of great importance as diagnostic and therapeutic agents. In a eukaryotic cell environment, glycosylation is attached to a secreted or membrane-spanning protein by co- and post-translational modification. Proteins destined for the cell surface are first co-translationally translocated into the lumen of the endoplasmic reticulum (ER) mediated by a signal sequence at or near the amino terminus of the nascent chain. Inside the ER, the signal sequence is usually removed and a high-mannose core oligosaccharide unit is attached to the asparagine (N) residue(s) present as part of the sequence Asn-X-Ser/Thr, where X is any amino acid except, perhaps, proline.
The efficiency of this co-translational glycosylation step is dependent on the presentation of an appropriate conformation of the peptide chain as it enters the endoplasmic reticulum (Imperiali and O""Connor, Pure and Applied Chem., 70: 33-40 (1998)). Potential N-linked glycosylation sites may no longer be accessible after the protein has folded (Komfeld and Komfeld, Ann Rev. Biochem. 54:631-664 (1985)). Proteins next move from the ER to the Golgi apparatus where further modifications, such as sulfation and processing of the high-mannose oligosaccharide chain to a complex-type oligosaccharide, occur and the proteins are directed to their proper destinations.
N-linked oligosaccharides can have a profound impact on the pharmaceutical properties of glycoprotein therapeutics (e.g., in vivo half-life and bioactivity). Different bioprocess parameters (e.g., bioreactor type, pH, media composition, and ammonia) have been shown to affect protein glycosylation significantly. Changes in terminal glycosylation (sialylation and galactosylation) and N-glycan branching are the most frequently observed alterations.
Tissue plasminogen activator (t-PA), a glycoprotein, is a multidomain serine protease whose physiological role is to convert plasminogen to plasmin, and thus to initiate or accelerate the process of fibrinolysis. Initial clinical interest in t-PA was raised because of its relatively high activity in the presence, as compared to the absence, of fibrin. Wild-type t-PA is a poor enzyme in the absence of fibrin, but the presence of fibrin strikingly enhances its ability to activate plasminogen. Recombinant human t-PA is used therapeutically as a fibrinolytic agent in the treatment of acute myocardial infarction and pulmonary embolism, both conditions usually resulting from an obstruction of a blood vessel by a fibrin-containing thrombus.
In addition to its striking fibrin specificity, t-PA exhibits several further distinguishing characteristics:
(a) T-PA differs from most serine proteases in that the single-chain form of the molecule has appreciable enzymatic activity. Toward some small substrates, and toward plasminogen in the absence of fibrin, two-chain t-PA has greater activity than one-chain t-PA. In the presence of fibrin, however, the two forms of t-PA are equally active (Rijken et al., J. Biol. Chem. 257: 2920-2925 (1982); Lijnen et al., Thromb Haemost., 64: 61-68 (1990); Bennett et al., J. Biol. Chem., 266: 5191-5201 (1991)). Most other serine proteases exist as zymogens and require proteolytic cleavage to a two-chain form to release full enzymatic activity.
(b) The action of t-PA in vivo and in vitro can be inhibited by a serpin, PAI-1 (Vaughan et al., J. Clin. Invest., 84: 586-591 (1989); Wiman et al., J. Biol. Chem., 259: 3644-3647 (1984)).
(c) T-PA binds to fibrin in vitro with a Kd in the xcexcM range.
(d) T-PA has a rapid in vivo clearance that is mediated by one or more receptors in the liver (Nilsson et al., Thromb. Res., 39: 511-521 (1985); Bugelski et al., Throm. Res., 53: 287-303 (1989); Morton et al., J. Biol. Chem., 264: 7228-7235 (1989)).
A substantially pure form of t-PA was first produced from a natural source and tested for in vivo activity by Collen et al., U.S. Pat. No. 4,752,603 issued Jun. 21, 1988 (see also Rijken et al., J. Biol. Chem., 256: 7035 (1981)). Pennica et al. (Nature, 301: 214(1983)) determined the DNA sequence of t-PA and deduced the amino acid sequence from this DNA sequence (U.S. Pat. No. 4,766,075 issued Aug. 23, 1988).
Human wild-type t-PA has potential N-linked glycosylation sites at amino acid positions 117,184,218, and 448. Recombinant human t-PA (ACTIVASE(copyright) t-PA) produced by expression in CHO cells was reported to contain approximately 7% by weight of carbohydrate, consisting of a high-mannose oligosaccharide at position 117, and complex oligosaccharides at Asn-184 and Asn-448 (Vehar et al., xe2x80x9cCharacterization Studies of Human Tissue Plasminogen Activator produced by Recombinant DNA Technology, xe2x80x9d Cold Spring Harbor Symposia on Quantitative Biology, LI:551-562 (1986)).
Position 218 has not been found to be glycosylated in native t-PA or recombinant wild-type t-PA. Sites 117 and 448 appear always to be glycosylated, while site 184 is thought to be glycosylated only in a fraction of the molecules. The t-PA molecules that are glycosylated at position 184 are termed Type I t-PA, and the molecules that are not glycosylated at position 184 are termed Type II t-PA. In melanoma-derived t-PA, the ratio of Type I to Type II t-PA is about 1:1. The most comprehensive analysis of the carbohydrate structures of CHO cell-derived human t-PA was carried out by Spellman et al., J. Biol. Chem. 264: 14100-14111 (1989), who showed that at least 17 different Asn-linked carbohydrate structures could be detected on the protein. These ranged from the high-mannose structures at position 117 to di-, tri-, and tetra-antennary N-acetyllactosamine-type structures at positions 184 and 448. Type I and Type II t-PAs were reported to be N-glycosylated in an identical way at Asn-117 and Asn-448 positions, when isolated from the same cell line. For further details, see also Parekh et al., Biochemistry, 28: 7644-7662 (1989). The specific fibrinolytic activity of Type II t-PA has been shown to be about 50% greater than that of Type I t-PA (Einarsson et al., Biochim. Biophys. Acta, 830: 1-10 (1985)). Further, increased Type I is correlated with increased half-life (Cole et al., Fibrinolysis, 7: 15-22 (1993)). However, Type II t-PA, which lacks a portion of carbohydrate associated with Type I t-PA, as well as desialated t-PA, demonstrated a longer Txc2xd beta than standard t-PA (Beebe and Aronson, Thromb. Res. 51: 11-22 (1988)).
Analysis of the sequence of t-PA has identified the molecule as having five domains. Each domain has been defined with reference to homologous structural or functional regions in other proteins such as trypsin, chymotrypsin, plasminogen, prothrombin, fibronectin, and epidermal growth factor (EGF). These domains have been designated, starting at the N-terminus of the amino acid sequence of t-PA, as the finger (F) domain from amino acid 1 to about amino acid 44, the growth factor (G) domain from about amino acid 45 to about amino acid 91 (based on homology with EGF), the kringle-1 (K1) domain from about amino acid 92 to about amino acid 173, the kringle-2 (K2) domain from about amino acid 180 to about amino acid 261, and the serine protease (P) domain from about amino acid 264 to the carboxyl terminus at amino acid 527. These domains are situated essentially adjacent to each other, and are connected by short xe2x80x9clinkerxe2x80x9d regions. These linker regions bring the total number of amino acids of the mature polypeptide to 527, although three additional residues (Gly-Ala-Arg) are occasionally found at the amino terminus. This additional tripeptide is generally thought to be the result of incomplete precursor processing, and it is not known to impart functionality. Native t-PA can be cleaved between position 275 and position 276 (located in the serine protease domain) to generate the two-chain form of the molecule.
Each domain contributes in a different way to the overall biologically significant properties of the t-PA molecule. Domain deletion studies show that the loss of the finger, growth factor, or kringle-2 domain results in a lower-affinity binding of the variant t-PA to fibrin (van Zonneveld et al., Proc. Natl. Acad. Sci. USA, 83: 4670-4674 (1986); Verheijen et al., EMBO J., 5: 3525-3530 (1986)); however, more recent results obtained with substitution mutants indicate that the kringle-2 domain is less involved in fibrin binding than earlier expected (Bennett et al., supra). The domain deletion studies have implicated the finger and growth factor domains in clearance by the liver (Collen et al., Blood, 71: 216-219 (1988); Kalyan et al., J. Biol. Chem., 263: 3971-3978 (1988); Fu et al., Thromb. Res., 50: 33-41 (1988); Refino et al., Fibrinolysis, 2: 30 (1988); Larsen et al., Blood, 73: 1842-1850 (1989); Browne et al., J. Biol. Chem., 263: 1599-1602 (1988)). The kringle-2 domain is responsible for binding to lysine. The serine protease domain is responsible for the enzymatic activity of t-PA and contains specific regions where mutations were shown to affect both fibrin binding and fibrin specificity (possibly direct interactions with fibrin), and other regions where only fibrin specificity is altered (possibly indirect interactions with fibrin) (Bennett et al., supra) . Studies with mutants resulting from site-directed alterations indicate the involvement of the glycosylation of t-PA in clearance (Lau et al., Bio/Technology, 5: 953-958 (1987); Lau et al., Bio/Technology, 6: 734 (1988)).
Several reports have suggested that the carbohydrated moueties of t-PA influence the in vitro activity of this enzyme (Einarsson et al., supra; Opdenakker et al., Proc. Sci. Exp. Biol. Med., 182: 248-257 (1986)). T-PA is endocytosed by mannose receptors of liver endothelial cells and by galactose receptors of parenchymal cells. Indeed, the in vivo clearance of recombinant human t-PA produced in mammalian cell cultures was influenced by carbohydrate structures, particularly by the high-mannose oligosaccharides (Hotchkiss et al., supra). At-PA variant (designated TNK t-PA) that has a glycosylation site added at amino acid position 103, the native glycosylation site removed at amino acid position 117, and the sequence at amino acid positions 296-299 of native human t-PA replaced by AAAA, has been shown to have increased circulatory half-life, and markedly better fibrin specificity than wild-type human t-PA (Keyt et al, Proc. Natl. Acad. Sci. USA, 91: 3670-3674 (1994)).
Cells expressing tPA-6, a molecule composed of the kringle-2 and serine protease domains of t-PA, process it into two glycoforms, a monoglycosylated form with Asn-448 occupied, and a diglycosylated form with Asn-448 and Asnl84 occupied (Berg et al., Blood, 81: 1312-1322 (1993)).
Plasminogen exists in two glycoforms. The more glycosylated form, commonly referred to as xe2x80x9cplasminogen-1,xe2x80x9d xe2x80x9cplasminogen I,xe2x80x9d or xe2x80x9cType 1 plasminogen,xe2x80x9d has a galactosamine-based oligosaccharide attached at amino acid position 345 (Thr345) and a complex glycosamine-based oligosaccharide at amino acid position 288 (Asn288) of a native human plasminogen molecule. The less glycosylated form, commonly referred to as xe2x80x9cplasminogen-2,xe2x80x9d xe2x80x9cplasminogen II,xe2x80x9dor xe2x80x9cType 2 plasminogen,xe2x80x9d has a single oligosaccharide chain attached at amino acid position 345 (Thr345) (Hayes and Castellino, J. Biol. Chem., 254(18): 8772-8776, 8777-8780 (1979); Lijnen et al., Eur. J. Biochem., 120: 149-154 (1981); Takada et al., Thrombosis Research, 39: 289-296 (1985)).
Other glycoproteins displaying variable site occupancy (variations in N- and O-glycosylation site-occupancy) include granulocyte-macrophage colony-stimulating factor (Okamoto et al, Archives of Biochemistry and Biophysics, 286: 562-568(1991)), interferon-gamma(Curlingetal, Biochem. J.,272: 333-337(1990)),protein C (Miletich and Broze, J. Biol. Chem. 265: 11397-11404 (1990)), and interleukin-2. Glycosylation of gamma-interferon was stable throughout an optimized culture design strategy using fed-batch cultures, with exposure to glucose starvation possibly leading to a dramatic change in glycosylation efficiency (Xie et al, Biotechnol. Bioeng., 56: 577-582 (1997)).
Different factors have been discussed to be potentially responsible for variable site-occupancy, including availability of dolichol-phosphate and nucleotide sugars (Nyberg et al., Biotechnol. Bioeng., 62: 336-347 (1999)), glycosyltransferase activity (Hendrickson and Imperiali, Biochemistry, 34: 9444-9450 (1995); Kaufman et al., Biochemistry, 33: 9813-9819 (1994)), and variable glycosylation site accessibility due to competition with protein folding (Holst et al., The EMBO J., 15: 3538-3546 (1996); Imperiali, Acc. Chem. Res., 30: 452-459 (1997); Shelikoff et al., Biotechnol. Bioeng., 50: 73-90 (1996)). Any of these factors could be influenced by cell culture conditions. T-PA site-occupancy usually varies within a rather narrow range (xc2x15%).
Asparagine-linked glycosylation involves the enzyme-catalyzed modification of an asparagine side chain in a nascent polypeptide with a tri-antennary tetradeca-saccharide moiety. This first committed step in the biosynthesis of N-linked glycoproteins is catalyzed by oligosaccharyltransferase, a heteromeric membrane-associated enzyme complex found in the lumen of the endoplasmic reticulum of eukaryotic cells. See Imperiali, supra; Allen et al., J. Biol. Chem., 270:4797-4804 (1995); Sharma et al., Eur. J. Biochem., 116:101-108 (1981); Silberstei and Gilmore, The FASEB Journal, 10: 849-858 (1996); Kumar et al., Biochem. Mol. Biol. Intl., 36: 817-826 (1995) Bause et al., Biochem. J., 312: 979-985 (1995); Xu and Cowardi Biochemistry, 36: 14683-14689 (1997); Kumar et al., Biochem. Biophys. Res. Comm. 247: 524-529 (1998); Watt et al., Curr. Op. Struct. Biol., 7: 652-660 (1997
For optimal activity, oligosaccharyltransferase requires a small amount of manganese divalent ion, but other divalent metal cations with an octahedral coordination geometry will support transfer, although at reduced rates (Hendrickson and Imperiali, supra; Kaufman et al., supra; Kumar et al., Biochem. and Mol. Biol. International, 36: 817-826 (1995)).
To simulate normal body environment, fermentor temperature in cultivating mammalian cells is controlled almost exclusively at 37xc2x0 C. This dogma is so widely accepted that, so far, little attention has been paid to varying temperature in the cell culture process. The scarce literature data suggest that reduced fermentor temperature results in improved cell viability and shear resistance, higher cell density and titer in batch cultures, and a reduction in glucose/lactate metabolism (Chuppa et al., Biotechnol. Bioeng., 55: 328-338 (1997)).
Specifically, Reuveny et al., J. Immunol. Methods, 86: 53-59 (1986) studied the effect of temperatures in the range of 28xc2x0 C. to 37xc2x0 C. on batch hybridoma cell cultures. They found that although at lower temperatures the cell viability was improved, this was accompanied by a decrease in glucose uptake and a decrease in the specific antibody production. Therefore, in this particular case, lower temperatures did not enhance the overall performance of the cell culture process.
Sureshkumar and Mutharasan, Biotechnol. Bioeng., 37: 292-295 (1991) investigated the effect of the temperature range of 29xc2x0 C. to 42xc2x0 C. on the cell culture process, and found that maximum cell density was achieved at 33xc2x0 C. In contrast, the glucose uptake and specific lactate production rates were dramatically lower at 33xc2x0 C. than at 39xc2x0 C. These results showed that the optimal temperatures for growth and productivity may considerably differ. While the viability increase at temperatures below 37xc2x0 C. appears to be a general phenomenon, the effect of temperature on specific productivity has been shown to be cell-line dependent (Chuppa et al., supra).
Weidemann et al, Cytotechnology, 15: 111-116 (1994) cultivated adherent recombinant baby hamster kidney (BHK) cells at temperatures between 30xc2x0 and 37xc2x0 C. The low-temperature cultivation in batch and repeated batch mode in a two-liter bioreactor showed a lower growth rate and a lower glucose consumption rate (i.e., less lactate production). On the other hand, the maximum cell density and productivity were not affected by the temperature reduction.
Kretzmer et al., xe2x80x9cCultivation Temperaturexe2x80x94Effect on Cell Culture Processes and Their Optimizationxe2x80x9d (American Chemical Society Meeting, San Francisco, Calif.), abstract 138, presented Apr. 16, 1997, disclosed the effect of cultivation temperature on cell culture processes and their optimization, but apparently no specific glycosylation analysis.
It has been suggested that reduced fermentor temperatures might have other advantages related to product quality and integrity, but the effect of low temperatures on product quality, and in particular, on protein glycosylation, has been scarcely studied. Chuppa et al., supra, have reported that fermentation temperature did not have a significant effect on the sialic acid content of glycoproteins. Although the total sugar content was somewhat lower at 37xc2x0 C. than at 34xc2x0 C. or 35.5xc2x0 C., the authors viewed this difference as xe2x80x9cnot substantial.xe2x80x9d
However, U.S. Pat. No. 5,705,364 described preparing glycoproteins by mammalian cell culture wherein the sialic acid content of the glycoprotein produced was controlled over a broad range of values by manipulating the cell culture environment, including the temperature. The host cell was cultured in a production phase of the culture by adding an alkanoic acid or salt thereof to the culture at a certain concentration range, maintaining the osmolality of the culture at about 250 to about 600 mOsm, and maintaining the temperature of the culture between about 30 and 35xc2x0 C.
Bahr-Davidson, xe2x80x9cFactors Affecting Glycosylation Site Occupancy of ASN- 184 of Tissue-Type Plasminogen Activator Produced in Chinese Hamster Ovary Cells,xe2x80x9d A Dissertation submitted to the Department of Chemical Engineering and the Committee of Graduate Studies of Stanford University in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy, May 1995, investigated the effects of temperature on glycosylation site occupancy and reported that site occupancy was increased by exposing cells to 26xc2x0 C. (see pages 50-51).
The effect of various additives such as components of plasma to the culture media on protein production and glycosylation has been studied in the literature, for example, the effects of hormonal treatments on membrane glycosylation in rat kidney brush broder membranes (Mittal et al., Indian J. Exp er. Biol., 34: 782-785 (1996)). Studies of Muc-1 mucin expression established the hormonal basis for mRNA expression (Parry et al., J. Cell Sci., 101: 191-199 (1992)). Thyroid hormone regulation of alpha-lactalbumin with differential glycosylation has been reported (Ziska et al., Endocrinology, 123: 2242-2248 (1988)). The cellular response to protein N-glycosylation was increased in the presence of thyroxine, insulin, and thrombin, and the effect was dose-dependent (Oliveira and Banerjee, J. Cell. Physiol., 144: 467-472 (1990)). Thyroxine was found to induce changes in the glycosylation pattern of rat alpha-fetoprotein (Naval et al., Int. J. Biochem. 18: 115-122 (1986)).
In addition to hormonal treatments, glutathione and glucose-6-phosphate dehydrogenase deficiency increased protein glycosylation (Jain, Free Radical Biology and Medicine, 24: 197-201 (1998)). Thyrotropin was found to control oligosaccharyltransferase activity in thyroid cells (Desruisseau et al., Mol. Cell. Endocrinol., 122: 223-228 (1996)). The addition of glucose and tri-iodothyronine (T3) to a medium producing a pro-urokinase derivative improved productivity (Hosoi et al., Cytotechnology, 19: 1-10 (1996)). Also, fucosyltransferase activity in the rat small intestine was responsive to hydrocortisone regulation during the suckling period (Biol et al., Biochim. Biophys. Acta, 1133: 206-212 (1992)). Hydrocortisone treatment also induced quantitative alterations in glycosylation of mouse mammary tumor virus precursors (Maldarelli and Yagi, JNCI, 77: 1109-1115 (1986)). Glycosylation of cellular glycoconjugates in a carcinoma cell line was enhanced by a retinoic acid (Sacks et al., Glycoconjugate J., 13: 791-796 (1996)). Further, retinoic acid had reversible effects on glycosaminoglycan synthesis during differentiation of HL-60 leukemia cells (Reiss et al., Can. Res., 45: 2092-2097 (1985)). Additionally, retinoic acid, as well as hydrocortisone, was found to modulate glycosaminoglycan synthesis of human malignant keratinocytes (Reiss et al., J. Invest. Dermatol., 86: 683-688 (1986)).
The competition between folding and glycosylation in the endoplasmic reticulum has been described (Holst et al., supra), as has acute heat shock inducing the phenomenon of prompt glycosylation (Jethmalani et al., J. Biol. Chem., 269: 23603-23609 (1994)).
There is a need for increasing glycosylation site occupancy in glycoproteins having multiple glycoforms to produce glycoprotein therapeutics of consistent product quality. For example, there is a need to increase the fraction of Type I t-PA in the t-PA production process. Such increase in site-occupancy generates t-PA with activity more closely resembling the international human t-PA standard, and thus more closely resembling human t-PA. Type I t-PA is also more soluble than Type II, which may be of some value in processing steps. Further, increased Type I is correlated with increasing circulatory half-life, as noted above.
It has been found that during the production of a wild-type glycoprotein, namely human t-PA, in mammalian cells, namely Chinese Hamster Ovary (CHO) cells, use of certain divalent metals, hormones, or factors that manipulate cell-cycle distribution to control or influence glycosylation significantly increases site occupancy of a glycosylation site of the glycoprotein. For example, decreasing the cultivation temperature from 37xc2x0 C. to about 30-35xc2x0 C. in the production phase significantly enhances the occupancy of the glycosylation site at amino acid position 184, and thereby increases the ratio of Type I t-PA to Type II t-PA. Specifically, decreasing the temperature from 37 to 33 or 31xc2x0 C. increased t-PA site-occupancy up to 6%. Temperatures below 37xc2x0 C. are expected similarly to facilitate the occupancy of not-easily-accessible N-linked glycosylation sites in other glycoproteins. Accordingly, temperature can be used as a sensitive tool for fine tuning the ratio of variously glycosylated forms of glycoproteins having one or more N-linked glycosylation sites occupied only in a fraction of the protein.
In addition, other environmental factors, including those that manipulate the culture""s growth state, and correspondingly cell-cycle distribution, such as butyrate, or a cell-cycle inhibitor that increases the proportion of cells in the G0/G1 phase such as quinidine, plasma components such as thyroid hormones, and/or certain divalent metal cations significantly elevated the t-PA Type I content (about 1-2.5%) compared to control conditions, and are expected to act similarly with respect to other glycoproteins. In contrast, addition of the relevant nucleoside precursor molecules (e.g., uridine, guanosine, mannose) did not result in improved site-occupancy.
In one aspect, the invention concerns a process for producing a glycoprotein comprising culturing mammalian host cells producing the glycoprotein (i.e., cells expressing nucleic acid encoding the glycoprotein) in the presence of (a) a factor that modifies growth state in a cell culture, (b) a divalent metal cation that can adopt and prefers an octahedral coordination geometry, or (c) a plasma component, whereby the occupancy of an N-linked glycosylation site occupied only in a fraction of the glycoprotein is enhanced in the glycoprotein so produced. Preferably, the factor is a cell-cycle inhibitor that blocks cells in the G0/G1 phase, a butyrate salt, and/or a temperature of the culture of between about 30 and 35xc2x0 C., the divalent cation is manganese or iron, and the plasma component is a hormone. Preferably, the cell culture procedure includes a growth phase, followed by a transition phase and a production phase. In a preferred embodiment, in the growth phase the mammalian host cells are cultured at about 37xc2x0 C., whereupon, during the transition phase, the temperature is lowered to between about 30xc2x0 C. and 35xc2x0 C. The host cells preferably are CHO cells, and the glycoprotein preferably is t-PA.
In another aspect, the invention provides a process for producing human t-PA comprising culturing CHO cells expressing nucleic acid encoding said t-PA in a serum-free medium in a production phase at a temperature of between about 30xc2x0 C. and 35xc2x0 C. and in the presence of about 0 to 2 mM of a butyrate salt, whereby the occupancy of an N-linked glycosylation site occupied only in a fraction of t-PA is enhanced in the t-PA so produced.
In a still further aspect, the invention supplies a process for producing human t-PA comprising culturing CHO cells expressing nucleic acid encoding said t-PA in a serum-free medium in a growth phase at a temperature of about 37-40xc2x0 C., wherein said medium comprises from about 10 xcexcM to 100 xcexcM of a divalent metal cation that can adopt and prefers an octahedral coordination geometry; culturing said cell in a transition phase at a temperature of about 37-40xc2x0 C.; and culturing said cell in a production phase wherein after about 48 hours into the production phase the temperature is lowered to between about 30xc2x0 C. and 35xc2x0 C. and about 0.75 to 1.5 mM of a butyrate salt is ad to the medium, whereby the occupancy of an N-linked glycosylation site occupied only in a fraction of t-PA is enhanced in the t-PA so produced. In this process, a plasma component such as a thyroid hormone, e.g., thyroxine or tri-iodothyronine, or a cell-cycle inhibitor that blocks cells in the G0/G1 phase such as quinidine is optionally added to the culture medium before or during the growth phase.
Hence, the process herein facilitates the production of a preferred glycoform of a glycoprotein, such as Type I t-PA, in a mammalian cell culture, and also increases the ratio of preferred to non-preferred glycoproteins, J such as the ratio of Type I to Type II t-PA, in a mammalian cell culture.